Many types of animal cells attach to and proliferate on various types of permeable surfaces such as collagen membranes. Attached to a thin transparent membrane, masses of cells can grow in a histophysiologic way, obtaining the required metabolites and disposing of products of metabolism by diffusion across the permeable supporting membrane. Under these conditions of culture, both columnar and stratified epithelial tissues are polarized. Initiation of cellular renewal in the tissue takes place near the plane of attachment to the membrane, and all metabolic exchange with the medium outside the pouch occurs across the membrane. These conditions are "histophysiologic". Histophysiologic gradient cultures mimic the situation in nature where attachment and exchange of metabolites occur at the stromal-parenchymal interface. The apparatus of the present invention provides a setting for cell growth similar to that found in nature.
In the history of studying tumor growth by transplantation to animals, a number of sites, some as chambers, have been employed. Examples are the anterior chamber of the guinea pig and the frog eye, the hamster cheek pouch, and subcutaneous sites when an injection of air and inoculum establish a pouch. Inocula of tumors in each of these examples are in an extremely complex setting, since in each case the biology of the host is poorly understood. The vitelline membrane enclosing the yolk of the chick egg has been used as a wrapping, a pouch, for the growth in culture of human gastrointestinal carcinomas.
Considerable societal pressure exists to reduce the use of laboratory animals in toxicology. There are two important questions to consider in such testing, the presence and degree of toxic effect and the potential for reversal of the toxic effect. Tests where death of tissue is the end point do not provide information on reversibility or recovery. If an organoid association of cells displays as its first response to a toxic material a consistent disruption of architectural organization, such a disruption may or may not be reversed by the prompt removal of the toxic material. Models of the type described here, using test cells such as human cell lines or primary isolates of human amnion or epidermis, may provide useful alternatives to laboratory animals.
The motivation to develop kits for preparation of histophysiologic gradient cultures are two-fold. First, there is a growing appreciation today in cell biology research of the physiologic importance of cells in organoid tissue arrangements. The configuration of cells as tissue is significant in areas as diverse as hepatic function, and physiologic response of mammary carcinoma cells to therapy. A recent study even suggests that the spatial relationship between cancer cells appears to determine the rapid acquisition of multicellular drug resistance to alkylating agents (Graham, C. H., et al., J.N.C.I. (1994) 86:975-982).
Second, the development of a histophysiologic gradient culture system will accelerate the acquisition of fundamental information and the study of problems of clinical oncology.
Various systems have been devised for culturing adherent cells supported on permeable membranes. A review of some of these methods is found in Leighton, J Cell Biochem (1994) 56:29-36. These methods include collagen-coated cellulose sponges, encapsulation into microcapsules, and, less conveniently, animal-derived sites such as the anterior chamber of guinea pig and frog eye, the hamster cheek pouch. Use of animal-derived environments has obvious disadvantages, and the various methods described in the prior art lack convenience and simplicity of construction.